Hi, there. I have cloned my target gene in to a lentivirus expression vector (pCDH-EF1α-MCS*-T2A-GFP, SBI CD526A-1). Is it possible to convert the T2A seqeunce to IRES?
Hi Zheng,
If you want to replace the T2A for IRES you'll also have to add an stop codon for your cloned gen.
Do you have the vector map and sequence? You could check whether you have restriction sites to remove the T2A (without cutting the ORF you cloned or the GFP in the vector) and amplify the IRES including the stop codon for your ORF and ends matching, or complementary, to the insertion site you used to remove the T2A (for regular ligation) or with overhangs matching the resulting backbone (for ligase independent Gibson assembly).
Cheers,
Sounds like a headache. These GC and structure rich sequences are difficult to clone. I think you'd be best off ordering the exact construct you want from a synthesis company.
Yes, you can swap out the 2A peptide sequence with an internal ribosomal entry site (IRES), but as mentioned above, one would need to introduce a stop codon for the newly incorporated IRES.
While the encephalomyocarditis virus (EMCV) IRES is GC-rich, I have PCR amplified and subcloned multiple versions of this IRES sequence without difficulty by using an engineered family B polymerase such as Phusion or Kapa HiFi and T5 flap endonuclease mediated isothermal assembly (aka Gibson assembly). Sanger sequence the final construct to ensure no mutations were PCR introduced (recommended not to skip this step).
Depending on your experimental design, you should plan to introduce the preferred IRES (viral bases 273-845; rather than the minimal IRES viral bases 400-836). You should also be familiar with the EMCV IRES A6 or A7 bifurcation loop. Ann Palmenberg's group explains the bias between the upstream or downstream cistron regarding the bifurcation loop and provides an illustration for clarity, so please review the paper:
Bochkov YA, Palmenberg AC. Translational efficiency of EMCV IRES in bicistronic vectors is dependent upon IRES sequence and gene location. Biotechniques. 2006 Sep;41(3):283-4, 286, 288 passim. PubMed PMID: 16989088.
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